Breast Cancer Risk-associated Snps In The MTOR Promoter Form De Novo KLF5 And ZEB1 Binding Sites That Influence The Cellular Response To Paclitaxel

Objective:The mTOR gene is located at chromosome 1q36.2.mTOR acts as a central switch that determines cell fate in tumorigenesis,including cell proliferation,migration,and multidrug resistance.The SNP rs2295080?-141G/T?,located in the promoter of the mTOR gene,has been found to be associated with an increased risk of various cancers.Given the functional importance of rs2295080,this SNP warrants further investigation to determine how it is controlled by key transcriptional factors,whether SNP rs2295080 located in linkage haplotypes with other SNPs,and the further potential functional impacts on cell proliferation,progression and drug response in breast cancer.Breast cancer is a leading cause of cancer-related death in women,with an estimate of more than two million new cases worldwide in 2018,and it accounts for 30%of all new cancer cases in women in the United States.Accumulating evidence supported that the hereditary genetic variations of candidate genes,such as single nucleotide polymorphisms?SNPs?and their haplotypes,induced inter-individualized cancer susceptibility and diverse responses to chemotherapeutic agents.Compared to conventional analysis of individual single SNPs,recently developed multi-allelic haplotype-based association analysis based on a cluster of 2–3 SNP markers significantly improved the power and robustness of detecting cancer susceptibility loci and responsiveness variability.It is important to understood why SNPs form haplotypes,and the functional advantages the linked SNPs have,and what the underlying molecular mechanisms are,need to be further examined.Methods:1 Study populationAll subjects gave their written informed consent prior to inclusion in the ongoing study.547 female patients with histopathologically confirmed breast cancer and 504 female healthy volunteers were consecutively recruited from First Hospital of China Medical University between October 2008 and June 2013.After an interview,5 ml of peripheral venous blood was collected for further SNP genotyping.Clinical data were obtained from the interviewer-administered health risk questionnaires and medical records.The healthy controls were recruited from individuals who visited the same hospitals for a health check-up,had no known medical illness or hereditary disorders and were not taking any medications.2 SNP genotypingGenomic DNA was isolated from a leukocyte cell pellet of each blood sample by using previously described methods.The TaqMan allelic discrimination method was used to genotype the selected SNPs.The SNP allele-specific probes were labeled with the fluorescent dyes HEX and FAM by using the TaqMan SNP Genotyping Assays of the ABI 7500 Fast Real-Time PCR platform.3 ImmunohistochemistryThe samples were incubated overnight in 4?with the primary antibody.The immunostaining was examined under a light microscope by two pathologists blinded to the experimental conditions.The intensity of immunoreactivity was scored as follows:0 for no staining,1 for weak staining,2 for moderate staining,and 3 for strong staining.The percentage of stained cells was scored by using 5%increments?0%,5%,10%,….100%?.The final immunoreactive score was determined by multiplying the intensity score with the percentage of positively stained cells.4 Western-BlotThe total protein of MCF7 or MCF7/PTX cells was extracted and quantified.Sixty micrograms of proteins from each cell lysate was separated by 10%SDS-PAGE electrophoresis and transferred to a nitrocellulose membrane.Primary antibodies were incubated overnight at 4?.The secondary antibody was incubated at room temperature for 1 h.Chemiluminescence was detected by ECL.5 ImmunofluorescenceMCF7 cells were seeded at a density of 5x10⁴ cells/well in a chamber slide.Twenty-four hours after transfection,cells were fixed with 4%?w/v?paraformaldehyde in PBS for 10 min.Primary antibodies were incubated overnight at 4?.Fluorescence was detected using a confocal laser scanning microscope.6 Cell-cycle analysisMCF7 and MCF7/PTX cells were cultured in 6-well plates.After transfection,the cells were cultured with 7.3 n M PTX for another 24 h.Cells were harvested,and cell density was adjusted to 10⁷ cells/ml.The cell cycle was analyzed by flow cytometry.The percentage of cells in S phase in the different groups were compared.The experiment was repeated at least three times.7 In vivo xenograft experimentsThe transplanted tumor model of BALB/c nude mice was prepared by subcutaneous injection at the scapula of MCF7 cells.Every 3 days,the weights of the xenograft mice were recorded until the end of the experiment.Ten days after cell injection,tumor formation was measured at intervals of 3 days.At the same time,PTX was injected through the caudal vein at the first time in the groups of PTX alone,pcmTOR-Ht1 and pcmTOR-Ht2 treated,once every three days a total of 6 times.Simultaneously,the NC group was established?n=5?.The effects on the weight of the xenograft mice and in vivo tumor growth were observed.The plasma was collected 4h after PTX injection at20,26 and 32 days.Mice were sacrificed and tumors were weighed and harvested for histological examination and UPLC-MS/MS.8 UPLC-MS/MS assayTissue from tumors was weighed and then homogenized.Cells were cultured in 6-well plates.pcmTOR-Ht1,pcmTOR-Ht2 and NC were transfected into each group.Twenty-four hours later,the cells were cultured with 7.3n M PTX for another 24 h.Then,cells were lysed by repeated freezing and thawing.After centrifugation at10,000×g for 5 min,supernatant was collected for UPLC-MS/MS analysis.The detail procedure was shown in the Supplemental methods.9 Luciferase reporter assayThe cells were transferred to 96-well plates.The pGL3-Ht1,pGL3-Ht2 and pRL-TK plasmids were transfected into 293T or MCF7 cells.FLuc luminescence was measured by using Dual-Luciferase?Reporter Assay System.The value of Fluc luciferase activity was corrected by Rluc luciferase activity.Relative luciferase activity was normalized to the negative control.Assays were conducted in triplicate in a single experiment,and then as three independent experiments.10 Fusion protein-Nano LUC luciferase reporter analysisThe pNLuc-ZEB1 and p NLuc-KLF5 could generate an in-frame Nluc fusion protein.The pGL3-Ht1,pGL3-Ht2,pNLuc-ZEB1 and pNLuc-KLF5 plasmids were transfected at a concentration of 1?g/ml.Cells were harvested at the following times:4,8,12,16,20,and 24 h,and then luciferase activity was detected.11 Chromatin immunoprecipitationThe plasmids pcmTOR-Ht1,pcmTOR-Ht2 were transfected to MCF7 cells.Samples were collected and subjected to immunoprecipitation with ZEB1 antibody and KLF5antibodyovernight at 4?.DNA fragments were purified by Genomic DNA Extraction Kit.Two microliters of CHIP extraction was subjected to qPCR assay.PCR products were sequenced.Results:1 Chemotherapy response and prognosis associated polymorphism and haplotype identification in the mTOR promoter regionWe discovered that rs2295080?-141G/T?and rs2295079?-78C/G?constitute linkage haplotypes,with Ht1?-78C/-141G?and Ht2?-78G/-141T?being dominant.Further cancer risk analysis revealed that Ht2 was significantly associated with an increased susceptibility of breast cancer?adjusted OR?95%CI?:1.339?1.096-1.637?,P=0.004?.The distribution frequencies of Ht1 and Ht2 were significantly associated with tumor size?P=0.019?,clinical stages?P=0.004?,and lymph node metastasis status?P=0.015?.Multivariate Cox regression analysis repeated and verified that the haplotypes?Ht2 vs.Ht1?were independent prognostic indicators for both OS and DFS in breast cancer patients,and Ht2 is associated with a significant shorter OS?P=0.008,adjusted HR?95%CI?:2.535?2.339-3.847??and DFS?P=0.010,2.224?2.072-3.695??than Ht1.2 mTOR haplotypes are associated with the expression of mTOR in breast cancer patient tissuesThe imbalanced expression and prognosis data indicate that Ht2 is associated with expression differences in local genes and exhibits a strong prognostic impact on survival in breast cancer patients who received NCT,likely through altering the expression of mTOR.3 Prioritizing mTOR haplotypes for functional assays in human breast cancer cellsAssays of cell viability in MCF7 cells showed that the proliferation of time course pcmTOR-Ht2 was much higher than pcmTOR-Ht1 in MCF7 but not in MCF7/PTX cells.In PTX-sensitive MCF7 cells,forced expression of pcmTOR-Ht2 substantially increased PTX IC₅₀ from 4.17 nM?control?to 61.61 nM,but there was no significant difference in MCF7/PTX cells.The intracellular PTX concentration was significantly reduced in MCF7 cells transfected with pcmTOR-Ht2.We observed that the percentage of cells in S phase among MCF7 cells transfected with pcmTOR-Ht2 was significantly elevated compared with those transfected with pcmTOR-Ht1 or NC,but there was no significant difference in MCF7/PTX cells.4 In vivo effects of mTOR haplotypes on tumor growth and chemoresistance of human breast cancer in xenograft miceFurthermore,IHC assays showed that the levels of p-mTOR,mTOR,ABCB1 and CCND1 were much higher in tumors with Ht2 overexpression than with Ht1.The cellular PTX concentration of the pcmTOR-Ht2+PTX group was significantly decreased in comparison to the pcmTOR-Ht1+PTX and NC+PTX groups.However,there was no significant difference observed in the PTX concentration of the plasma samples.These above data indicate that,compared to Ht1,Ht2 exhibits a stronger effect on promoting expression of mTOR,which in turn results in a much more robust effect on promoting tumor growth and conferring resistance to PTX treatment in vivo.5 Transcription factor binding analysesAssays of luciferase activities showed that the transcriptional activities of Ht2 sequence were much higher than those of Ht1 sequence in both 293T and MCF7 cell lines?P<0.0001?.We verified that transcriptional activities of pGL3-Ht1 can be continuously and time-dependently decreased by KLF5?P<0.0001?,indicating that KLF5 acts as an orientation-independent transcriptional silencer.In contrast,pGL3-Ht2 with ZEB1 had relatively higher target gene promoter activity than without ZEB1?P<0.0001?,suggesting that ZEB1 acts as an orientation-dependent enhancer.6 Prioritizing candidate haplotypes for bioinformatics and functional assaysCompared with pcmTOR-Ht1,mRNA and protein levels of p-mTOR,m TOR,ABCB1and CCND1 in MCF7 transfected with pcmTOR-Ht2 were increased.Taken together,the competition with transcription factor binding sites suggested that the haplotypes synergistically contribute to substantially promoting the expression of mTOR by facilitating the binding of ZEB1?an enhancer?and inhibiting the binding of KLF5?a silencer?Conclusions:In the present study,a haplotype based case-control was performed,and SNPs in the 5'UTR of mTOR gene were genotyped and identified.We discovered that rs2295080?-141G/T?and rs2295079?-78C/G?constitute linkage haplotypes,with Ht1?-78C/-141G?and Ht2?-78G/-141T?being dominant,which are associated with distinct susceptibility to breast cancer,response to PTX and clinical prognosis.We further found that Ht1 and Ht2 have silencer/enhancer properties,and a novel ZEB1 binding site created by the-141T allele of Ht2 and an emerging KLF5 binding site that exists on the-78C allele of Ht1 synergistically affect expression of mTOR in response to diverse PTX and clinical outcomes.Mechanistically,ZEB1/KLF5 exerts its transcription regulation by direct binding with allele of mTOR haplotypes,thereby promote/inhibit mTOR gene expression,cell proliferation and excretion of cytotoxic drugs through the ZEB1/KLF5-mTOR-CCND1/ABCB1 cascade,thereby affecting the response to PTX treatment.